Biological applications of Pyoverdines and their complexes with Iron and Gallium
Eliana Schlosser-Silverman, Marcia Ben-Shoshan, Shlomo Pleban*, Dorit Zharhary
Biotechnology R&D Department, Sigma-Aldrich Israel
Abstract
Pyoverdines (pseudobactins) are fluorescent siderophores that have highaffinity for iron (10-32M). The fluorescent Pseudomonas group synthesizes
them under iron-deficient growth conditions. Pyoverdines are composed of
three structural parts: a dihydroxyquinoline chromophore, a side chain and a
variable peptidic chain. The peptide moiety is involved in receptor recognition
and binding. The size and amino acid composition of pyoverdines are unique
to each species, as well as the pyoverdine recognition specificity. Despite this
specificity, many fluorescent pseudomonads are able to utilize pyoverdines
produced by other strains.
We have isolated three different siderophores of the pyoverdine type from
Pseudomonas fluorescens. These pyoverdines, differing in their side chain,
were identified by HPLC and mass spectra (m/z 1161, 1190 and 1160). We
show that these three forms of pyoverdines, when complexed to iron, are
effective growth promoters for bacteria in a minimal medium. In contrast, a
mixture of these pyoverdines complexed to Gallium acts as a useful inhibitor
of growth for both Pseudomonas fluorescens and Pseudomonas aeruginosa.
In addition, these pyoverdines are effective in protecting Escherichia coli from
the deleterious effects of reactive oxygen intermediates (ROI). These results
shed light on novel uses of pyoverdines and their complexes as ROI protecting
and antibacterial agents.
Pv
Pv
basic pH acidic pH
Fe-Pv
Ga-Pv
Fermentation Broth
Titration to Acidic pH
Bind to FeCl3.6H2O (Fe-Pv) or Ga(NO3)3. H2O (Ga-Pv)
Chromatographic purification using gel filtration
Fermentation and Purification
Purified Compounds: Pv; Fe-Pv; Ga-Pv
250C
300C
Figure 3. Purification of Pyoverdines (Pv) and their Iron and Gallium complexes
Component Identification
P. fluorescens was grown at 25ºC (left) and 30ºC (right).
Only at 25ºC Pyoverdines were produced (fluorescent green).
Figure 1. Pyoverdines production is temperature dependent
Side chain
m/z
-NH-CO-CH2-CH2-COOH
Succinic acid
1161
-NH-CO-CH2-CH2-CHOH-CONH2
2-hydroxy glutaramide
1190
-NH-CO-CH2-CH2-CONH2
Succinamide
1160
Components of the pyoverdines mixture.
Each component is composed of three structural parts: Peptide chain, Chromophore
and Side chain.
The three components of the mixture differ in their side chain.
Figure 4. Pyoverdines mixture components
P. Fluorescens 5 liter fermentation
The green color is indicative of pyoverdines production
This fermentation was also performed in large scale fermentors
Figure 2. Pyoverdines fermentation
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1160
400
B
A
400
control
1190
300
3.5
SNG
200
pyo
200
1000
3
100
pyo + SNG
00
20
40
60
80
20
40
60
2.5
OD 600
-50
00
-100
80
1.5
75
1
50
0.5
25
100
2
10
1
control
0
0
-20
00
20
40
60
80
SNG
Streptonigrin (SNG) antibiotic activity is iron-activated. The DNA cleavage
reaction and chromosome damage by SNG are influenced by the nature of
the metal ion present and dependent on the production of free radicals.
1161
800
Pyo+SNG
0.1
Time (minutes)
100
Pyo
50 100 150 200 250 300 350
400
200
-100
00
20
40
60
80 2.0
Purified Pyoverdines mixture (center) and the three purified pyoverdines components
of the mixture
Figure 5. HPLC chromatograms
E. coli was grown in LB to early log phase. Free Pyoverdines mixture (pyo)
was added at 19 mM (A) or 38 mM (B). 20 minutes after the addition of
pyo, the indicated cultures were treated with Streptonigrin (Sigma code
S1014 )(SNG) 1.5 mg/ml (A), or 1.25 mg/ml (B). Samples were taken at
different time points for absorbance measurement (A) or at 150 minutes
for viable counts (B).
Figure 8. Pyoverdines protect against Streptonigrin-induced DNA damage
Biological Activity
B
A
untreated
1.2
OD 600
1
untreated P.f
Fe-Pv 1
Fe-Pv P.f
Fe-Pv 2
Fe-Pv 3
0.8
0.6
0.4
• Three pyoverdine compounds were purified from P. fluorescens
fermentations, differing in their side chain: the succinic acid form
(1161), the succinamide (1160) and the 2-hydroxy glutaramide form
(1190).
1.8
1.6
1.4
OD 600
1.4
Summary
1.2
1
0.8
0.6
• All three forms were active as growth promoters of P. fluorescens when
complexed to iron.
0.4
0.2
0
0.2
0
0
2
4
6
0
8
1
2
3
4
5
6
7
8
9
Time (hours)
Time (hours)
P. fluorescens was grown in iron depleted minimal medium without or
with the addition of (A) a mixture of 0.14 mM Fe-Pv from P. fluorescens or
with (B) the addition of Fe-Pv 0.8 mM components:
Fe-Pv 1: 2-hydroxy glutaramide (1190); Fe-Pv 2: Succnic acid (1161); Fe-Pv
3: Succinamide (1160)
Figure 6. A mixture of Ferri-pyoverdines (Fe-Pv) and its three components promote
P. fluorescens growth
Possible Applications of Pyoverdines
• Prevention of iron overload toxicity
• Inhibition of pathogenic bacterial growth (as iron chelator).
1.4
untreated P.f
untreated P.a
1.2
Ga-PV P.f
Ga-Pv P.a
The Following Products are Available from Sigma:
1
OD 600
OD 600
• Pyoverdines protect E. coli from the deleterious effects of Streptonigrin,
possibly by its iron chelating activity. This chelating activity also leads to
E. coli inhibition of growth.
• Specific inhibitor of fluorescent pseudomonads strains (when complexed
to Gallium)
B
A
0.5
0.45
0.4
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0
• The complex Gallium-Pyoverdines acts as a useful growth inhibitor for
both P. fluorescens and P. aeruginosa, suggesting that Ga-Pyoverdines
can be used as specific antimicrobial agents.
0.8
• P8374 Pyoverdine Iron complex (Fe-Pv)
0.6
• P8249 Pyoverdines Gallium (Ga-Pv)
0.4
0.2
• P8124 Pyoverdines (Pv)
0
0
2
4
Time (hours)
6
8
0
2
4
6
8
Time (hours)
P. fluorescens (A) and P. aeruginosa (B) were grown in iron depleted minimal
medium without or with the addition of 0.7 mM Ga-Pv from P. fluorescens
Figure 7. Gallium-pyoverdines (Ga-Pv) inhibit P. fluorescens and P. aeruginosa
growth
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